**2.4 Microbial characterization and identification in the biofiltration system**

DNA samples of the microbial consortium along different lengths of the biofilter were extracted using an Easy-DNATM Kit (Invitrogen, USA) following the manufacturer's instructions. The 16S rRNA gene was amplified using universal bacterial primers. Polymerase chain reaction (PCR) was conducted as previously reported (Garcia-Peña et al., 2011). The PCR amplification products were purified using a QIAquick PCR Purification Kit (Qiagen, UK). The PCR-amplified DNA products were separated by DGGE on 8% polyacrylamide gels with a linear gradient of 5–30% denaturant (100% denaturant was 40% [v/v] formamide plus 42% [w/v] urea) using the DCodeTM System (Bio-Rad, Hercules, CA, USA). After purification, the PCR products were sent to a sequencing service (Macrogen Inc., Korea). The nucleotide sequences of each 16S rRNA gene were aligned using the Clustal X software (Higgins et al., 1996). Identification of the sequences was made after performing BLAST searches of the NCBI database.
